LDH cytotoxicity assay

Cell cytotoxicity was assessed by measuring the release of cytoplasmic lactate dehydrogenase (LDH) into cell culture supernatants according to the manufacturer's protocol (CytoTox 96 Non-Radioactive Cytotoxicity Assay, Promega, Madison, WI). For TDLN B cell cytotoxicity, effector B cells were generated from 4T1 TDLN B cells using LPS/anti-CD40 as described above. Target cells were plated in triplicates in a 96-well U-bottom tissue culture plate (5000 cells/well) and co-incubated with TDLN B cells at effector to target cell ratios of 1:1, 3:1, 10:1 and 30:1. After 12 hours of incubation, cells were centrifuged and 50 μl supernatant from each well was transferred to a fresh 96-well plate, 50 μl of the substrate mix was added and incubated at room temperature in the dark for 15 to 30 min. Before LDH measurement, 50 μl of stop solution was added to each well. Maximal release of LDH was performed by incubating the target cells with Lysis Solution (Promega, Madison, WI). Target cells without effector cells were used as a spontaneous release control. Absorbance was measured at 490 nm using a 96-well plate reader.